Antiviral antibiotic BU-4224V

ABSTRACT

A novel antibiotic complex designated BU-4224V produced by fermentation of Kibdelosporangium albatum sp. nov. Strain R761-7. The complex may be separated chromatographically into bioactive components designated BU-4224V A, B 1 , B 2 , and C. The components BU-4224V B 1  and B 2  display both antiviral and antimicrobial activity, while component BU-4224V A has antimicrobial activity and component BU-4224V C has antiviral activity.

This application is a division, of application Ser. No. 07/559,864,filed Jul. 27, 1990 U.S. Pat. No. 5,256,646.

BACKGROUND OF THE INVENTION

The present invention relates to a novel antibiotic complex havingantiviral and/or antimicrobial activity, to its production, recovery andseparation into four bioactive components, and to a pharmaceuticalcomposition thereof.

SUMMARY OF THE INVENTION

There is provided by the present invention a new antibiotic complexdesignated BU-4224V, said complex being produced by cultivating aBU-4224V-producing strain of Kibdelosporangium albatum, preferablystrain Kibdelosporangium albatum, sp. nov. strain R761-7, or variants ormutants thereof, in an aqueous fermentation culture nutrient mediumcontaining assimilable sources of nitrogen and carbon under submergedaerobic conditions until a substantial amount of the antiviralantibiotic complex BU-4224V is produced by the organism in thefermentation culture nutrient medium and subsequently, recovering theBU-4224V complex from the culture medium. The BU-4224V complex containsfour bioactive components designated BU-4224V A, BU-4224V B₁, BU-4224VB₂, and BU-4224V C which may be separated by conventionalchromatographic procedures.

The BU-4224V A, B₁, B₂, and C components exhibit antiviral activityagainst herpes simplex virus type 1 (HSV-1) in the dye uptake assay andthe plaque reduction assay, and/or antimicrobial activity in the agardilution method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HPLC chromatograms of the BU-4224V components.

FIG. 2 shows the IR spectra of BU-4224V B₁.

FIG. 3 shows the IR spectra of BU-4224V B₂.

FIG. 4 shows the ¹ H-NMR spectra of BU-4224V B₁.

FIG. 5 shows the ¹³ C-NMR spectra of BU-4224V B₁.

FIG. 6 shows the ¹ H-NMR spectra of BU-4224V B₂.

FIG. 7 shows the ¹³ C-NMR spectra of BU-4224V B₂.

FIG. 8 shows the structures of BU-4224V B₁ and B₂.

FIG. 9 shows the IR spectra of BU-4224V A.

FIG. 10 shows the IR spectra of BU-4224V C.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel antiviral and/or antimicrobialantibiotics designated herein as BU-4224V A, B₁, B₂, and C and to theirpreparation by fermentation of certain strains of Kibdelosporangiumalbatum, most particularly Kibdelosporangium albatum sp. nov. StrainR761-7.

Strain R761-7 was isolated from a soil sample collected in MindanaoIsland, the Philippines. A biologically pure culture of this strain wasdeposited with the American Type Culture Collection, Rockville, Md.under the accession number ATCC 55061.

The cultural and physiological characteristics of the strain wereexamined by the methods of Shirling & Gottlieb¹ and Shearer et al.²Diagnostic amino acid and sugar in the whole cell hydrolysate wereanalyzed by the methods of Lechevalier.³ The phospholipids wereidentified by the descriptions of Lechevalier et al.⁴ The menaquinonesamples were prepared by the procedures of Collins et al,⁵ and analyzedwith a mass spectrometer. The detection of mycolate and the glycolatetest were carried out by the methods of Minnikin et al,⁶ and Uchida &Aida,⁷ respectively.

The Microorganism

The actinomycete strain R761-7 which produces antiviral and/orantimicrobial antibiotics BU-4224V A, B₁, B², and C was isolated from asoil sample collected in the Philippines. The morphology, cultural andphysiological characteristics and chemotaxonomy of strain R761-7indicated that the strain is classified into the genusKibdelosporangium. Based on the direct or descriptive comparisons of thestrain to known species of the genus, the strain was designatedKibdelosporangium albatum sp. nov.

Morphology

Strain R761-7 is an aerobic, gram-positive, non-acid-fast filamentousorganism that forms well-branched substrate and aerial mycelia. Thesubstrate mycelium exhibits varying degrees of fragmentation. The aerialmycelium bears long straight chains of spores and many sporangiumlikeglobular bodies (8˜20 μm in diameter). The spores are cylindrical(0.4×0.8˜2 μm), and have smooth surface without distinct sheath. Thesporangiumlike globules with membranous envelope contain irregularlycurved, branched hyphae, but not spores, and have rugose surface.Germination occurs directly from these globules. Motile cells are notobserved.

Cultural characteristics

Aerial mycelium is formed on most agar media, and is white or yellowishwhite in mass-color. The substrate mycelium is colorless to yellowishbrown or orange yellow. Melanin and other distinct pigments are notproduced. The cultural characteristics are shown in Table 1.

Physiological characteristics

Gelatin and potato starch are hydrolyzed. Milk is coagulated andpeptonized. NaCl tolerance is seen at 4% but not at 5% or more. Thegrowth occurs between 17° C. and 45° C. No growth is observed in 14° C.and 48° C. The physiological characteristics are shown in Table 2.

Chemotaxonomy

Whole cell hydrolysate contains meso-diaminopimelic acid as thediagnostic amino acid. Whole cell-sugar consisted of rhamnose, ribose,arabinose, glucose and galactose. Madurose is not detected. Thephospholipids contain two phosphatidylethanolamines,phosphatidylglycerol and phosphatidylinositol. Thus, strain R761-7 has atype IV cell wall with a sugar pattern A, and a type P-II phospholipid.The major menaquinone is MK-9 (H₄). Mycolate is absent. Glycolate testis negative (N-acyl type of peptidoglycan: acetyl).

Taxonomic position

Based on the micro-morphological study by photomicroscopy and scanningelectron microscopy, the sporangiumlike structure of strain R761-7 isdifferentiated from the sporangium of Streptosporangium andSpirillospora, from the pseudosporangium of Actinosporangium andActinomadura, and also from the sclerotium of Chainia.

The chemotaxonomy of strain R761-7 revealed that the cell chemistry ofstrain R761-7 is closely related to genera Amycolatopsis⁸ andKibdelsporangium² but is differentiated from the other hither-todescribed, spore-forming genera of actinomycetes. The morphology andchemotaxonomy of strain R761-7 is consistent with those of the genusKibdelosporangium; that is Kibdelosporangium is characterized by theformations of hyphi-enveloping sporangiumlike structure as well as sporechain on the aerial mycelium, and by the type IV-A cell wall, type P-IIphospholipids, MK-9 (H₄) major menaquinone and lack of mycolate. StrainR761-7 is also similar to the genus in the cultural characteristics,e.g. the formation of white aerial mycelia and the moderate or goodgrowth both on the natural organic media and chemically defined media.

Thus, strain R761-7 was classified into the genus Kibdelosporangium,which includes only two species and one subspecies, (E. aridum,²,9 K.philippinense,¹⁰ and K. aridum subsp. largum¹¹). The direct ordescriptive comparison of the characteristics of strain R761-7 to thoseof the two known species of genus Kibdelosporangium are shown in Table2. Strain R761-7 is different from all known species ofKibdelosporangium in the lack of melanin formation in any of ISP medianos. 1, 6 and 7, the hydrolysis of starch, the acid formation fromadonitol, and the lack of acid formation from D-melezitose, melibioseand α-methyl-D-glucoside.

The comparative considerations between strain R761-7 and two knownspecies of Kibdelosporangium led us to classify this strain as a newspecies of the genus. Hence, the designation, Kibdelosporangium albatumsp. nov. is proposed for the strain. The type strain is No. R761-7 whichis single isolate.

                                      TABLE 1                                     __________________________________________________________________________    Cultural characteristics of strain R761-7                                     Medium        Growth                                                                              Aerial mycelium                                                                          Substrate mycelium                                                                        Diffusible pigment                 __________________________________________________________________________    Sucrose-nitrate agar                                                                        Good  Moderate; pale yellow                                                                    Moderate orange yellow                                                                    Light yellowish brown              (Czapek-Dox agar)   (89)       (71)        (76)                               Tryptone-yeast extract broth                                                                Moderate,                                                                           None       Light yellowish brown                                                                     None                               (ISP No. 1)   pellicle,        (76)                                                         not turbid                                                      Yeast extract-malt                                                                          Good  Poor; white                                                                              Deep yellow (85)                                                                          None                               extract agar (ISP No. 2)                                                      Oatmeal agar (ISP No. 3)                                                                    Moderate                                                                            Poor; white to light                                                                     Colorless to light                                                                        None                                                   yellow (86)                                                                              brown (57)                                     Inorganic salts-starch agar                                                                 Good  Moderate; yellowish                                                                      Moderate yellowish                                                                        None                               (ISP No. 4)         white (92) brown (77)                                     Glycerol-asparagine agar                                                                    Moderate                                                                            Moderate; yellowish                                                                      Brilliant orange                                                                          None                               (ISP No. 5)         white (92) yellow (67)                                    Peptone-yeast extract-                                                                      Moderate                                                                            Moderate; white                                                                          Vivid yellow (82)                                                                         Pale yellow (89)                   iron agar (ISP No. 6)                                                         Tyrosine agar (ISP No. 7)                                                                   Moderate                                                                            Moderate; white                                                                          Brilliant orange                                                                          Light orange                                                      yellow (67) yellow (70)                        Glucose-asparagine agar                                                                     Poor  Poor; white                                                                              Pale yellow (89)                                                                          None                               __________________________________________________________________________     Observation after incubation at 28° C. for 3 weeks                     Color name, used: ISCCNBS colorname charts.                              

                  TABLE 2                                                         ______________________________________                                        Physiological characteristics of strain R761-7 in comparison                  with two species of genus Kibdelosporangium                                              Strain    K.      K. phili-                                                   R761-7    aridum  ppinense                                         ______________________________________                                        Acid from:                                                                    Adonitol     +           -       -                                            L-Arabinose  +           +       -                                            D-Cellobiose +           +       +                                            Dextrin      +           +       -                                            Dulcitol     -           -       -                                            i-Erythritol -           -       -                                            D-Fructose   +           +       +                                            D-Galactose  +           +       +                                            D-Glucose    +           +       +                                            Glycerol     +           +       +                                            i-Inositol   +           +       +                                            Lactose        +(w)      +       +                                            Maltose      +           +       +                                            D-Mannitol   +           +       +                                            D-Mannose    +           +       +                                            D-Melezitose -           +       +                                            Melibiose    -           +       +                                            α-Methyl-D-                                                                          -           +       +                                            glucoside                                                                     Raffinose    -           +       -                                            L-Rhamnose   +           +       +                                            D-Ribose     +           +       +                                            Salicin      +           v       -                                            D-Sorbitol   -           -       -                                            L-Sorbose    -           -       -                                            Sucrose      +           +       -                                            Trehalose    +           +       +                                            D-Xylose     +           +       +                                            Production of:                                                                Nitrate reductase                                                                          -           -       +                                            Catalase     +           +       +                                            Hydrogen sulfide                                                                           +           +       +                                            Melanin      -           +       +                                            Hydrolysis of:                                                                Potato starch                                                                              +           -       -                                            Gelatin      +           +       +                                            Utilization of:                                                               Benzoate     -           -       -                                            Citrate      -           +       +                                            Mucate       -           -       UD                                           Succinate    +           +       +                                            Tartrate     -           -       -                                            Decomposition of:                                                             Adenine      -           -       -                                            Casein       +           +       +                                            L-Tyrosine   +           +       +                                            Hypoxanthine -           -       +                                            Xanthine     -           -       -                                            Cellulose    -           -       -                                            Urea         +           +       +                                            Esculin      +           +       +                                            Hippurate    +           +       +                                            Growth at/in:                                                                 10° C.                                                                              -           +       -                                            15° C.                                                                              ±        +       -                                            42° C.                                                                              +           +       -                                            45° C.                                                                              -           Trace   -                                            Lysozyme broth                                                                               +(w)      -       +                                            4% NaCl      +           +       -                                            5˜7% NaCl                                                                            -           +       -                                            8% NaCl      -           -       -                                            ______________________________________                                         UD: undescribed                                                               (w): weak                                                                     K. aridum subsp. largum ATCC 39922 is reported to be not differentiated       physiologically from K. aridum ATCC 39323 (J. Antibiotics 39: 1386-1394,      1986).                                                                   

Fermentation

The BU-4224V antibiotics of the present invention are produced bycultivating a BU-4224V producing strain of Kibdelosporangium albatum,preferably Kibdelosporangium albatum, ATCC 55061, or a mutant or variantthereof, under submerged aerobic conditions in an aqueous nutrientmedium. The producing organism is grown in a nutrient medium containingan assimilable carbon source, for example an assimilable carbohydrate.Examples of suitable carbon sources include lactose, glycerol, sucrose,corn starch, glucose, mannose, fructose, cellobiose, trehalose, mannitoland xylose. The nutrient medium should also contain an assimilablenitrogen source such as fish meal, peptones, soybean meal, peanut meal,cotton seed meal and corn steep liquor. Nutrient inorganic salts mayalso be incorporated in the medium and such salts may comprise any ofthe usual salts capable of providing sodium, potassium, ammonium,calcium, phosphate, sulfate, chloride, bromide, nitrate, carbonate orlike ions.

Production of BU-4224V may be effected at any temperature conductive tosatisfactory growth of the organism, i.e., approximately 17°-45° C. andis conveniently carried out at a temperature of about 28° C. Ordinarily,optimum production is obtained after incubation period of about 4-6days. The fermentation may be carried out in flasks and in laboratory orindustrial fermentors of various capacities. When tank fermentation isto be carried out, it is desirable to produce a vegetative inoculum in anutrient broth by inoculating the broth culture with a slant or soilculture or a lyophilized culture of the organism. The medium in whichthe vegetative inoculum is produced can be the same as, or differentfrom, that utilized in the tank for the production of the new compoundsof the present invention as long as it is such that a good growth of themicroorganism is obtained.

Production of BU-4224V can be followed during the course of fermentationby testing samples of the broth or extracts of the mycelial solids foractivity against organisms known to be sensitive to the compounds of thepresent invention or by an in vitro cytotoxicity assay.

When fermentation is complete, the components are recovered from thefermentation broth and separated by extraction with a suitable organicsolvent followed by a series of column chromatographies. Example 2 belowillustrates specific procedures for obtaining the components A, B₁, B₂,and C. FIG. 1 shows the HPLC chromatograms of components A, B₁, B₂, andC.

As is the case with other microorganism, the characteristics of the newBU-4224V-producing strain of the present invention strain R761-7, (ATCC55061), are subject to variation. Recombinants, variants and mutants ofstrain R761-7 (ATCC 55061) may be obtained by treatment with variousknown mutagens such as ultraviolet rays, X-rays, high frequency waves,radioactive rays and chemicals. Natural and induced variants, mutantsand recombinants of strain R761-7, (ATCC 55061) which retain thecharacteristic of producing BU-4224V are intended to be encompassed bythe present invention.

Physico-chemical properties of BU.4224V B₁ and B₂

BU-4224V B₁ and B₂ were isolated as a white amorphous powder. They weresoluble in methanol, pyridine and dimethyl sulfoxide, slightly solublein ethyl acetate and acetone but practically insoluble in n-hexane,chloroform and water. They showed positive reactions to iodine andanthrone reagent but were negative to ninhydrin and Sakaguchi tests. Thephysico-chemical properties of components BU-4224V B₁ and B₂ aresummarized in Table 3. The UV spectra of BU-4224V B₁ and B₂ showed noabsorption maxima above 210 nm. The IR spectra of BU-4224V B₁ and B₂ areshown in FIGS. 2 and 3, respectively, and their ¹ H-and ¹³ C-NMR spectrain FIGS. 4-7.

Structure elucidation of BU-4224V B₁ and B₂

The molecular formulae of BU-4224V B₁ and B₂ were determined to be C₈₃H₁₅₂ O₃₃ and C₈₃ H₁₅₀ O₃₃, respectively, based on their elementalanalyses and negative fast-atom bombardment mass spectral data. The ¹H-NMR spectrum of B₁ showed the presence of five anomeric protons(δ5.23(2H), 4.89, 4.85 & 4.82), three 0-methyl groups (δ3.82(6H) &3.77), two C-methyl groups (δ1.34 and 1.22) and a large number ofC-methylene protons (δ1.1-1.9) (Table 4). The information together withthat of the IR and ¹³ C-NMR spectra suggested that BU-4224V B₁ wascomposed of sugars and long-chain fatty acids. The ¹ H-NMR spectrum ofBU-4224V B₂ was quite similar to that of BU-4224V B₁ except that adoublet of methyl signal (δ1.34) of the latter was absent and a singletmethyl (δ2.03) and a triplet methylene (δ2.36) signals were observed inthe former. Comparison of the ¹³ C-NMR spectra of B₁ and B₂substantiated the difference of carbon signals as deduced by the above ¹H-NMR (Table 5). The complete structures of BU-4224V B₁ and B₂ (FIG. 8),were elucidated by a combination of chemical degradation experiments andspectral analyses of the products. BU-4224V B₁ and B₂ containD-glucoses, 2-methoxy glucoses and hydroxylated long-chain fatty acids.

                  TABLE 3                                                         ______________________________________                                        Physico-chemical properties of BU-4224V B.sub.1 and B.sub.2                              BU-4224V B.sub.1                                                                          BU-4224V B.sub.2                                       ______________________________________                                        Nature       White amorphous                                                                             White amorphous                                                 powder        powder                                             M.p.         84-85° C.                                                                            82-83° C.                                   [α].sub.D.sup.26 (c 0.5, MeOH)                                                       -15.6° C.                                                                            -16.1° C.                                   (-)FAB--MS   m/z 1675 (M-1).sup.-                                                                        m/z 1673 (M-1).sup.-                               Mol. wt.     1676          1674                                               Elemental analysis                                                                         C.sub.83 H.sub.152 O.sub.33 .2H.sub.2 O                                                     C.sub.83 H.sub.150 O.sub.33 .2H.sub.2 O                           Calcd.  found       Calcd.                                                                              found                                           C   58.18   58.03   C   58.24 58.15                                           H    9.11    8.99   H    9.00  8.91                                IR (KBr)     3410, 2930, 2850                                                                            3430, 2930, 2850,                                  cm.sup.-1    1740, 1640, 1470                                                                            1735                                                            1370, 1200-1000                                                                             1720 (sh.), 1630,                                                             1470                                                                          1200-1000                                          TLC, SiO.sub.2                                                                             Rf 0.47       0.47                                               (EtOAc--MeOH--H.sub.2 O = 10:3:1)                                             TLC, RP-18   Rf 0.30       0.33                                               (Merck: CH.sub.3 CN-0.022M phosphate buffer, pH 7.0, 70:30)                   HPLC         Rt 8.2 min     9.2 min                                           (YMC-Pack D-ODS-5, CH.sub.3 CN-0.022M phosphate buffer,                       pH 7.0, 60:40)                                                                ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        .sup.1 H-NMR data of BU-4224V B.sub.1 and B.sub.2 (400 MHz,                   Pyridine-d.sub.5)                                                             BU-4224V B.sub.1 BU-4224V B.sub.2                                             ______________________________________                                        δ                                                                           5.23 (2H, br-d, J = 9.4)                                                                       δ                                                                             5.24 (2H, br-d, J = 9.8)                               4.89 (1H, d, J = 7.7)  4.90 (1H, d, J = 7.7)                                  4.85 (1H, d, J = 7.7)  4.85 (1H, d, J = 7.7)                                  4.82 (1H, d, J = 7.7)  4.82 (1H, d, J = 8.1)                                  4.4-4.6 (8H, m)        4.4-4.6 (8H, m)                                        4.34 (4H, m)           4.34 (4H, m)                                           4.15 (10H, m)          4.15 (10H, m)                                          3.9-4.1 (8H, m)        3.9-4.1 (7H, m)                                        3.86 (3H, m)           3.86 (3H, m)                                           3.82 (6H, s)           3.82 (6H, s)                                           3.77 (3H, s)           3.77 (3H, s)                                           3.62 (2H, dd, J = 4.7 &                                                                              3.62 (2H, dd, J = 4.3 & 15.4)                          15.4)                  3.42 (3H, q-like, J = 8.6)                             3.42 (3H, q-like, J = 8.7)                                                                           2.79 (2H, dd, J = 9.0 & 15.4)                          2.79 (2H, dd, J = 9.4 &                                                                              2.36 (2H, t, J = 7.3)                                  15.4)                  2.03 (3H, s)                                           1.1-1.9 (76H, m)       1.1-1.9 (74H, m)                                       1.34 (3H, d, J = 6.4)  1.22 (3H, d, J = 6.0)                                  1.22 (3H, d, J = 6.4)                                                     ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        .sup.13 C-NMR data of EU-4224V B.sub.1 and B.sub.2 (100 MHz,                  Pyridine-d5)                                                                  Carbon    BU-4224V B.sub.1                                                                           BU-4224V B.sub.2                                       ______________________________________                                         1        δ171.7                                                                             s     δ208.1                                                                            s                                         2        106.4      d     171.7     s                                         3        103.1      d     106.4     d                                         4        101.8      d     103.1     d                                         5        85.3       d     101.8     d                                         6        85.0       d     85.3      d                                         7        79.3       d     85.1      d                                         8        78.9       d     79.3      d                                         9        78.3       d     79.2      d                                        10        78.1       d     78.9      d                                        11        78.0       d     78.3      d                                        12        77.7       d     78.1      d                                        13        77.6       d     78.0      d                                        14        75.1       d     77.7      d                                        15        74.5       d     77.6      d                                        16        74.2       d     75.1      d                                        17        72.1       d     74.5      d                                        18        71.8       d     74.2      d                                        19        71.7       d     72.1      d                                        20        67.0       d     71.8      d                                        21        65.3       t     71.7      d                                        22        62.9       t     65.4      t                                        23        62.8       t     62.9      t                                        24        60.7       q     62.8      t                                        25        60.6       q     60.8      q                                        26        42.4       t     60.6      q                                        27        40.1       t     43.4      t                                        28        37.7       t     42.4      t                                        29        36.0       t     37.8      t                                        30        35.3       t     36.0      t                                        31        34.2       t     35.4      t                                        32        30.4       t     34.2      t                                        33        30.3       t     34.0      t                                        34        30.2       t     30.4      t                                        35        29.9       t     30.2      t                                        36        26.4       t     30.1      t                                        37        25.7       t     30.0      t                                        38        25.6       t     29.9      t                                        39        25.2       t     29.7      t                                        40        24.3       q     29.6      q                                        41        19.5       q     25.7      t                                        42                         25.6      t                                        43                         25.2      t                                        44                         24.9      t                                        45                         24.1      t                                        46                         19.5      q                                        ______________________________________                                         Physico-chemical properties of BU- 4224VA and C

The components BU-4224V A and C are minor products and found, by HPLC,to consist of two to four subcomponents. Their separation has not beencarried out and their structure has not been determined. Thephysico-chemical properties of BU-4224V A and C are summarized in Table6 and the IR spectra of BU-4224V A and C are given in FIGS. 9 and FIG.10, respectively. They have properties similar to those of the majorcomponents, BU-4224V B₁ and B₂.

                  TABLE 6                                                         ______________________________________                                        Physico-chemical properties of BU-4224V A and C                                          BU-4224V A  BU-4224V C                                             ______________________________________                                        Nature       White amorphous                                                                             White amorphous                                                 powder        powder                                             M.P.         77-78° C.                                                                            75-77° C.                                   [α].sub.D.sup.25 (c 0.5, MeOH)                                                       -13°   -20°                                        Negative FAB--MS                                                                           m/z 1743, 1683, 1647                                                                        m/z 1731                                           Elemental analysis                                                                         found (%)     found (%)                                                     C   46.02       C     57.66                                                   H    7.30       H      8.92                                        IR (KBr)     3410, 2930, 2850,                                                                           3400, 2920, 2850,                                  cm.sup.-1    1735          1735, 1640, 1470,                                               1720 (sh), 1640,                                                                            1360                                                            1460          1200-1000                                                       1360, 1200-1000                                                  UV (MeOH)      No absorption maxima above 210 nm                              TLC, SiO.sub.2 Rf 0.47     0.47                                               (EtOAc--MeOH--H.sub.2 O = 10:3:1)                                             TLC, RP-18   Rf 0.42       0.19                                               (Merck: CH.sub.3 CN-0.022M phosphate buffer, pH 7.0, 70:30)                   ______________________________________                                    

Antiviral Activity

Antiviral activity for each component of BU-4224V was evaluated by thedye uptake and the plaque reduction assay using herpes simplex virustype 1 (KOS strain) infection in Vero cells. In the dye uptake assay,200 μl of the Vero cell suspension containing 1.6×10⁴ cells was pouredinto each well of 96-well microplates, and then 50 μl of mediumcontaining a test compound at various concentrations was added to eachwell. The viral suspension (50 μl ) containing approximately 30X TCID₅₀was inoculated to each well. For cytotoxicity tests, the same set ofwells without viruses were prepared. After incubation at 37° C. for 72hours under the humidified 5% CO₂ -95% air environment, the degree ofinhibition of the virus-induced cytopathic effect and the drug-inducedcytotoxicity were determined by means of the uptake of neutral red. ID₅₀was expressed as the concentration showing the 50% inhibition of thecytopathic effect of control, and TD₅₀ was the concentration exhibitingthe 50% cytotoxicity against Vero cells without viral infection.Acyclovir was used as a reference compound of anti-HSV activity. Theantiviral activity of BU-4224V was also evaluated by the conventionalplaque reduction assay method using a 24-well microplate.

Each component of BU-4224V, except for component A, demonstrated apotent antiviral activity against HSV-1 with ID₅₀ values of 2˜5 μg/ml bythe dye uptake assay. This antiviral activity is less potent than thatof acyclovir. In the plaque reduction assay, BU-4224V showed almost thesame antiviral activity as in the dye uptake assay.

Among the BU-4224V components the component C showed most potentantiviral activity against herpes simplex virus type 1(HSV-1) in the dyeuptake assay and the plaque reduction assay. BU-4224V B₁ and B₂ werealmost at the same level in the above assays and 2-3 times less activethan BU-4224V C against HSV-1. Component C exhibited anti-HSV activitywith ID₅₀ values of 2.3 μg/ml by the dye uptake assay and 2.8 μg/ml bythe plaque reduction assay. All of the components exhibited nocytotoxicity (TD₅₀ :>400 mcg/ml, MDT: >200mcg/ml) against HSV-Verocells. A summary of the anti-HSV activity of each component of BU-4224Vis shown in Table 7.

Antimicrobial Activity

The antimicrobial spectra of BU-4224V B₁ and B₂ against various bacteriaand fungi are shown in Table 8. MICs were determined by the agardilution method using nutrient agar medium (Eiken) for aerobic bacteriaand Sabouraud dextrose agar for fungi. The inoculum size was adjusted to10³ -10⁴ cfu/ml for aerobic bacteria and 10⁶ for fungi.

BU-4224V B₁ and B₂ exhibited weak antibacterial activity against aerobicGram-positive bacteria and showed no activity against aerobicGram-negative bacteria and fungi.

Table 9 shows the antimicrobial activity of BU-4224V A and C. BU-4224V Aexhibited weak inhibitory activity only against Staphylococcus aureusFDA 209P, Smith, S. epidermidis D153 and Bacillus subtilis PCI219.BU-4224V C exhibited antimicrobial activity but not at 100 μg/ml.

                  TABLE 7                                                         ______________________________________                                        Anti-HSV activity of BU-4224V                                                        HSV-Vero cells                                                                Dye uptake assay                                                                            Plaque reduction assay                                          ID.sub.50                                                                             TD.sub.50 ID.sub.50 MTD*                                              (μg/ml)                                                                            (μg/ml)                                                                              (μg/ml)                                                                              (μg/ml)                                 ______________________________________                                        BU-4224V A                                                                             15.8      >400      44.1    >200                                     BU-4224V B.sub.1                                                                       4.9       >400      10.9    >200                                     BU-4224V B.sub.2                                                                       5.0       >400       9.3    >200                                     BU-4224V C                                                                             2.3       >400       2.8    >200                                     Acyclovir                                                                               0.09     >100       0.27   >100                                     ______________________________________                                         *MTD: Minimal toxic dose                                                 

                  TABLE 8                                                         ______________________________________                                        Antibacterial activity of BU-4224V B.sub.1 and B.sub.2                                         MIC (μg/ml)                                               Organism           BU-4224V B.sub.1                                                                         BU-4224V B.sub.2                                ______________________________________                                        Escherichia coli NIHJ                                                                            >100       >100                                            Klebsiella pneumoniae D11                                                                        >100       >100                                            Pseudomonas aeruginosa A9930                                                                     >100       >100                                            Proteus vulgaris A9436                                                                           >100       >100                                            Staphylococcus aureus FDA 209P                                                                      12.5      50                                            Staphylococcus aureus Smith                                                                         12.5    100                                             Staphylococcus aureus D136                                                                         25       >100                                            Staphylococcus aureus A15097                                                                       25       >100                                            Staphylococcus epidermidis D153                                                                      3.1        6.3                                         Streptococcus faecalis A9612                                                                     >100       >100                                            Micrococcus luteus 1001                                                                          >100       >100                                            Bacillus subtilis PCI219                                                                             3.1        6.3                                         Candida albicans IAM4888                                                                         >100       >100                                            Cryptococcus neoformans IAM4514                                                                  >100       >100                                            Aspergillus fumigatus IAM2034                                                                    >100       >100                                            Trichophyton mentagrophytes D155                                                                 >100       >100                                            ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Antimicrobial activity of BU-4224V A and C                                                     MIC (μg/ml)                                               Organism           BU-4224V A BU-4224V C                                      ______________________________________                                        Escherichia coli NIHJ                                                                            >100       >100                                            Klebsiella pneumoniae D11                                                                        >100       >100                                            Pseudomonas aeruginosa A9930                                                                     >100       >100                                            Proteus vulgaris A9436                                                                           >100       >100                                            Staphylococcus aureus FDA 209P                                                                     50       >100                                            Staphylococcus aureus Smith                                                                       100       >100                                            Staphylococcus aureus D136                                                                       >100       >100                                            Staphylococcus aureus A15097                                                                     >100       >100                                            Staphylococcus epidermidis D153                                                                     12.5    >100                                            Streptococcus faecalis A9612                                                                     >100       >100                                            Micrococcus luteus 1001                                                                          >100       >100                                            Bacillus subtilis PCI219                                                                             6.3    >100                                            Candida albicans IAM4888                                                                         >100       >100                                            Cryptococcus neoformans IAM4514                                                                  >100       >100                                            Aspergillus fumigatus IAM2034                                                                    >100       >100                                            Trichophyton mentagrophytes D155                                                                 >100       >100                                            ______________________________________                                    

According to one aspect of the invention, therefore, there is provided aprocess for the production of antibiotics by fermentation of aBU-4224V-producing strain of Kibdelosporangium albatum.

Another aspect of the invention provides a method for therapeuticallytreating animal and human viral infections by administering to theafflicted host an effective amount of BU-4224V B₁, B₂, or C, orcombinations thereof.

Another aspect of the invention provides a method for therapeuticallytreating animal and human microbial infections by administering to theafflicted host an effective amount of BU-4224V A, B₁, or B₂, orcombinations thereof.

In yet another aspect of this invention a pharmaceutical composition isprovided which comprises an effective amount of BU-4224V A, B₁, B₂, orC, or combinations thereof, in combination with an inert pharmaceuticalacceptable carrier or diluent. These compositions can be made up in anypharmaceutical form appropriate for the desired route of administration.

This invention also provides the microorganism Kibdelosporangium albatumhaving the identifying characteristics of strain No. R761-7, (ATCC55061).

The pharmaceutical composition provided by the present invention maycontain other active ingredients, e.g., other antibiotic agents, and maybe made up in any form appropriate for the desired route ofadministration. Examples of such compositions include solid compositionsfor oral administration such as capsules, tablets, pills, powders andgranules, liquid compositions for oral administration such as solutions,suspensions, syrups or elixirs and preparations for parenteraladministration such as sterile solutions, suspensions or emulsions. Theymay also be manufactured in the form of sterile solid compositions whichcan be dissolved in sterile water, physiological saline or other sterileinjectable medium immediately before use.

It will be appreciated that the actual dosages of the compounds of thepresent invention will vary according to the particular compound beingused, the particular composition formulated, the mode of administrationand the particular situs, host and disease being treated. Many factorsthat modify the action of the drug will be taken into account by thoseskilled in the art, e.g. age, body weight, sex, diet, time ofadministration, route of administration, rate of excretion, condition ofthe host, drug combinations, reactions sensitivities and severity of thedisease. Optimal dosages for a given set of conditions can beascertained by those skilled in the art using conventional dosagedetermination tests.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

References

1) Shirling, E. B. & D. Gottlieb: Methods for characterization ofStreptomyces species. Int. J. Syst Bacteriol. 16: 313-340, 1966

2) Shearer, M. C. et al.: New genus of the Actinomycetales:Kibdelosporangium aridum gen. nov., sp. nov. Int. J. Syst. Bacteriol.36: 47-54, 1986

3) Lechevalier, M.P.: Identification of aerobic actinomycetes ofclinical importance. J. Lab. Clin. Med. 71: 934-944, 1968

4) Lechevalier, M. P.; C. D. Bievre and H. Lechevalier: Chemotaxonomy ofaerobic actinomycetes: phospholipid composition. Biochem. Syst. Ecol. 5:249-260, 1977

5) Collins, M. D.; T. Pirouz, M. Goodfellow and D. E. Minnikin:Distribution of menaquinones in actinomycetes and corynebacteria. J.Gen. Microbiol. 100: 221-230, 1977

6) Minnikin, D. E.; L. Alshamaony and M. Goodfellow: Differentiation ofMycobacterium, Nocardia, and related taxa by thin-layer chromatographicanalysis of whole-organism methanolysates. J. Gen. Microbiol. 88:200-204, 1975

7) Uchida, K. and K. Aida: Taxonomic significance of cell-wall acyl typein Corynebacterium-Mycobacterium-Nocardia group by a glycolate test. J.Gen. Appl. Microbial. 25: 169-183, 1979

8) Lechevalier, M. P., H. Prauser, D. P. Labeda and J. S. Ruan: Two newgenera of nocardioform actinomycetes: Amycolata gen. nov. andAmycolatopsis gen. nov. Int. J. Syst. Bacteriol. 36: 29-37, 1986

9) Shearer, M. C. et al.: Aridicins, novel glycopeptide antibiotics. I.Taxonomy, production and biological activity. J. Antibiotics 38:555-560, 1985

10) Mertz, F. P. and R. C. Yao: Kibdelosporangium philippinense sp. nov.isolated from soil. Int. J. Syst. Bacteriol. 38: 282-286, 1988

11) Shearer, M. C. et al.: Kibdelins, novel glycopeptide antibiotics. I.Discovery, production, and biological evaluation. J. Antibiotics 39:1386-1394, 1986

EXAMPLE I Fermentation

A small agar piece of the slant culture of Kibdelosporangium albatumstrain R761-7 was inoculated into a 500-ml Erlenmeyer flask containing100 ml of the seed medium consisting of mashed potato 4%, corn steepliquor 2%, CaCO₃ 0.3% and NaCl 0.2% (the pH was adjusted to 8.0 beforeautoclaving). The seed culture was incubated at 28° C. for 4 days on arotary shaker (200 rpm) and 5 ml of the culture was transferred into a500-ml Erlenmeyer flask containing 100 ml of the production mediumhaving the same composition as the seed medium. The fermentation wascarried out at 28° C. for 6 days on a rotary shaker. The antibioticproduction in the fermentation broth was monitored by the conventionalcytopathic effect (CPE) assay method using herpes simplex virus type I(KOS strain). The production of the complex reached a maximum after 4 to5 days and the antiviral activity was observed x48 broth dilution interms of IC₅₀ value.

EXAMPLE II Isolation and purification

The fermentation broth (20 liters, 50-100 μg/ml) was stirred vigorouslywith n-butanol (8 liters) for 30 minutes. The n-butanol extract wasconcentrated in vacuo to dryness and the residue (9.0 g) was mixed withsilica gel (25 g) and loaded on the top of a silica gel column (WakogelC-200, 1.1 liters). The column was developed with ethylacetate-methanol-water (100:15:1) mixture. The eluate was collected infractions (20 ml) which were examined by the antiviral assay and TLC(SiO₂ ; EtOAc-MeOH-H₂ O, 10:3:1, H₂ SO₄ detection). Appropriatefractions were combined and concentrated in vacuo to give a crudemixture solid of BU-4224 V (1.0 g).

The solid (1.0 g) was dissolved in methanol (5 ml) and subjected toreversed phase C₁₈ column chromatography (YMC-ODS, AM type, YamamuraChem. Lab. Co. Ltd., 800 ml). The column was washed with a 0.022 Mphosphate buffer solution (pH 7.0) containing 45% CH₃ CN (1.5 liters)and then developed with the solution containing 50% CH₃ CN (fr. 1-65)and 55% CH₃ CN (fr. 66-98). Presence of the antibiotics was detected byTLC (RP-18, Merck: CH₃ CN-0.022M phosphate buffer, pH 7.0, 70:30).Fraction Nos. 9-15 (Rf. 0.42) were pooled, concentrated and extractedwith n-butanol. The n-butanol extract was evaporated in vacuo to afforda white powder of BU-4224V A (98 mg). Fraction Nos. 35-52 (Rf 0.32) andNos. 62-80 (Rf 0.19) were worked up by the same way to afford whitepowders of BU-4224V B (330 mg) and BU-4224 V C (140 mg), respectively.The minor components, BU-4224 V A and C were found to consist of 2 to 4subcomponents by HPLC (FIG. 1). Thus, the major component, BU-4224V Bwas also revealed to contain two components, BU-4224 V B₁ and B₂ (FIG.1). Separation of BU-4224V B mixture (140 mg) was carried out bypreparative HPLC (Column: YMC-Pack D-ODS-5, Yamamura Chem. Lab. Co.Ltd., 20×250 mm, mobile phase: CH₃ CN-0.01M phosphate buffer, pH 7.0,54:46, detection: UV 210 nm). The first peak cuts containing BU-4224 VB₁ were combined and concentrated. The solution was extracted withn-butanol and the extract was evaporated to a white powder of BU-4224 VB₁ (54 mg). This solid was chromatographed on a column of Sephadex LH-20(200 ml) eluting with 90% aqueous methanol to afford a pure sample ofBU-4224 V B₁ (48 mg). The second peak cuts containing component B₂ wereworked up by a similar manner to yield a pure solid of BU-4224 V B₂ (49mg).

We claim:
 1. The compound BU-4224 V A characterized as follows:(a)appearance: white amorphous powder (b) melting point: 77°-78° C. (c)optical rotation: [α]_(D) ²⁵ -13° (c 0.5, MeOH) (d) elemental analysis:C, 46.02%, H 7.30% (e) secondary ion mass spectrum: m/z 1743, 1683, 1647(f) IR (KBr): 3410, 2930, 2850, 1735, 1720(sh), 1640, 1460, 1360,1200-1000 (g) thin-layer chromatography: R_(f) =0.47, R_(f) =0.42(EtOAc-MeOH-H₂ O=10:3:1) (h) UV in MeOH: No absorption maxima above 210nm.
 2. The compound BU-4224V C characterized as follows:(a) appearance:white amorphous powder (b) melting point: 75°-77° C. (c) opticalrotation: [α]_(D) ²⁵ -20° (c 0.5, MeOH) (d) elemental analysis: C,57.66%, H 8.92% (e) secondary ion mass spectrum: m/z 1731 (f) IR (KBr):3400, 2920, 2850, 1735, 1640, 1470, 1360, 1200-1000 (g) thin-layerchromatography: R_(f) =0.47, R_(f) =0.19 (EtOAc-MeOH-H₂ O=10:3:1 ) (h)UV in MeOH: No absorption maxima above 210 nm.
 3. A method for treatinga mammalian host affected by herpes simplex virus type 1, which methodcomprises administering to said host an effective amount of the compoundof claim
 2. 4. A pharmaceutical composition comprising an effectiveamount of a compound of claim 1 or 2 or combinations thereof, togetherwith a pharmaceutical acceptable carrier.
 5. A method for treating amammalian host affected by bacterial infections, which method comprisesadministering to said host an effective amount of the compound of claim1.